The advantage of long reads in 16S rDNA-based microbiome studies

Mouse gut microbiome

16S rDNA-based species identification is the most commonly used method in microbiome studies1. Shin et al.2 examined the composition of mouse gut microbiota  using a 16S approach and either short-read sequencing technology or long-read nanopore sequencing.

The common use of the V3-V4 16S region for species identification is not as sensitive as using the full length of the 16S rRNA gene, as allowed by nanopore sequencing.

A key difference between the two approaches was that, for short-read experiments, the hypervariable V3-V4 16S region was used for primer design and amplicon generation (mean read length – 447 bp), while the full length of the 16S rRNA gene was used for the nanopore experiments (mean read length – 1393 bp).

Sequencing data derived from the two platforms were highly concordant (R2=0.8-0.9) at all taxonomic levels apart from the species level. It was shown that nanopore data allowed the identification of more species (i.e. Bifidobacterium animalis and Bifidobacterium pseudolongum, well-known members of the gut microbiome3), resulting in the construction of a fuller microbiota representation.

The study illustrates that the common use of the V3-V4 16S region for species identification is not as sensitive as using the full length of the 16S rRNA gene, as allowed by nanopore sequencing2.

In conclusion, nanopore sequencing’s long read advantage was instrumental for the generation of a more accurate profile of the mouse gut microbiome compared to short-read technology.

Fig 1.PNG

Figure: Variability in the 16S rRNA gene sequence. Detected variants between the 16S rRNA gene sequences are represented as vertical lines on the 16S rDNA  sequences. The black and red arrows indicate the binding positions of primer sets for the amplification of V3-V4 regions (for short-read sequencing) and nearly full-length regions of 16S rDNA sequences (nanopore sequencing), respectively. Image adapted from Shin et al.2

This case study is taken from the microbiome white paper.

1. Ranjan, R. et al. Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophys Res Commun. 469(4) (2016).

2. Shin, J. et al. Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing. Scientific Reports 6, 29681 (2016).

3. Turroni, F. et al. Bifidobacterium bifidum as an example of a specialized human gut commensal. Frontiers in Microbiology 5, 437